Thymidylate synthetase (TS) is the target enzyme for the chemotherapeutic drug 5-fluorodeoxyuridine (FdUrd). The goals of this project are to study the mechanisms by which cells become resistant to FdUrd and regulates TS activity during the cell cycle. A FdUrd-resistant 3T6 cell line has been isolated that overproduces TS and its mRNA by a factor of about 50 and regulate TS gene expression in the same manner as the parental 3T6 cells. Furthermore, cDNA corresponding to TS mRNA has been cloned. The specific aims for this grant period are first to obtain basic information about the structure of TS as well as its mRNA and its gene and second, to determine how the expression of the gene is regulated during the cell cycle. Mouse TS will be sequenced using a combination of protein and DNA sequencing procedures. The number of copies of the TS gene will be estimated to determine if overproduction of TS in the FdUrd-resistant cells is due to TS gene amplification. The structure of the normal and amplified genes will be studied to determine if the TS gene is rearranged in the drug-resistant cell line. The chromosome on which the mouse TS gene is located will also be determined. The mouse TS structural gene and adjacent DNA that is likely to contain transcriptional control sequences will be cloned and intervening sequences mapped. The regulation of TS gene expression during the cell cycle will also be studied. We will determine if TS mRNA production is regulated at the transcriptional level or by controlling the processing or export of TS sequences from the nucleus to the cytoplasm. The TS gene or appropriate derivatives will be cloned into a mammalian shuttle vector (derived from Bovine Papillomavirus). If transcriptional control during the cell cycle is still maintained, the location of the sequences responsible for this control will be studied, as will the specific proteins that may be associated with these control regions. The long range goal is to reconstruct control of TS gene transcription in an in vitro system and to determine if common regulatory proteins and DNA sequences are responsible for coordinating the expression of the genes for the S-phase enzyme during the cell cycle.